The present disclosure relates to composite microarray non-luminescent slides useful for carrying a microarray of biological polymers on the surface thereof and, more particularly, to composite microarray non-luminescent slides having a microporous membrane effectively attached by covalent bonding through a surface treatment to a substrate that prepares the substrate to sufficiently, covalently bond to the microporous membrane formed by a phase inversion process such that the combination produced thereby is useful in microarray applications and, most particularly, to composite microarray non-luminescent slides having a porous nylon membrane covalently bonded to a solid base member, such as, for example, a glass or Mylar microscope slide, such that the combination produced thereby is useful in microarray applications and to a process for producing such composite microarray non-luminescent slides.
A variety of methods is currently available for making arrays of biological macromolecules, such as, for example, arrays of nucleic acid molecules or proteins. One method for making ordered arrays of DNA on a porous membrane is a xe2x80x9cdot blotxe2x80x9d approach. In this method, a vacuum manifold transfers a plurality, e.g., 96, aqueous samples of DNA from three (3) millimeter diameter wells to a porous membrane. A common variant of this procedure is a xe2x80x9cslot-blotxe2x80x9d method in which the wells have highly-elongated oval shapes.
The DNA is immobilized on the porous membrane by baking the membrane or exposing it to UV radiation. This is a manual procedure practical for making one array at a timer and usually limited to 96 samples per array. xe2x80x9cDot-blotxe2x80x9d procedures are therefore inadequate for applications in which many thousand samples must be determined.
A more efficient technique employed for making ordered arrays of genomic fragments (e.g., PCR products) uses an array of pins dipped into the wells, e.g., the 96 wells of a microtitre plate, for transferring an array of samples to a substrate, such as a porous membrane. One array includes pins that are designed to spot a membrane in a staggered fashion, for creating an array of 9216 spots in a 22xc3x9722 cm area (Lehrach, et al., 1990). A limitation with this approach is that the volume of DNA spotted in each pixel of each array is highly variable. In addition, the number of arrays that can be made with each dipping is usually quite small.
Several patents have described the use of microarray slides in microarray applications. These include U.S. Pat. No. 5,919,626 entitled, xe2x80x9cAttachment of unmodified nucleic acids to silanized solid phase surfacesxe2x80x9d; U.S. Pat. No. 5,667,976 entitled, xe2x80x9cSolid supports for nucleic acid hybridization assaysxe2x80x9d and U.S. Pat. No. 5,760,130 entitled xe2x80x9cAminosilane/carbodiimide coupling of DNA to glass substratexe2x80x9d, the disclosure of each is herein incorporated by reference to the extent not inconsistent with the present disclosure.
Microarray slides are well known in the art. Schleicher and Schuell have attempted to attach nylon membrane to a glass slide using glue or similar adhesive in their commercially available CAST(trademark) slides. However, the layer of glue or adhesive adds additional thickness to the nylon membrane/glass slide combination, and the gluing/adhesive process may require the use of a scrim-reinforced nylon membrane. The extra thickness of the overall nylon membrane/glass slide combination caused by the glue/adhesive and the reinforcing scrim is a disadvantage in microarray applications. Additionally, the scrim makes the surface of the membrane of the nylon membrane/glass slide combination uneven and less than ideal from a cosmetic standpoint. Even further, the chemistry of the glue or adhesive used to attach the nylon membrane to the glass slide is not necessary optimal to effectuate the combination, nor is it necessarily compatible with the biomolecules or analytes for which the product is intended to receive, as it may interfere or react with the analyte.
Similarly, other products known to be currently commercially available include: Modified glass that binds nucleic acids or proteins without the use of a membrane; Corning GAPS Slides, such as, for example CMT-GAPS(trademark) coated slides; Nitrocellulose porous membrane cast onto glass, available from Schleicher and Schuell as FAST(trademark) Slides; Scrim-reinforced nylon glued or adhered to a glass substrate and Schleicher and Schuell CAST(trademark) Slides. Detailed descriptions of these commercially available products are readily available from the respective manufacturer and are known in the art.
However, in microarray applications, binding nucleic acids or proteins directly to a glass substrate has certain disadvantages. Specifically, a considerably smaller surface area for binding the nucleic acids or proteins is available than with a comparably sized microporous membrane/glass slide combination. The larger the binding surface area the, better the signal strength of the biomolecules or analytes, thereby allowing for the detection of smaller samples of biomolecules or analytes. Also, the porous membrane portion of the microporous membrane/glass slide combination naturally adsorbs the biomolecules or analytes and holds them in place on the microporous membrane/glass slide combination, whereas without the microporous membrane portion of the slide, the biomolecules or analytes would just sit on top of a glass surface, as there is no adsorption of the biomolecules or analytes. It is also likely that the efficiency of immobilization of biomolecule on the glass is substantially less than 100%, and may be less than 50%, when compared to immobilization of the target on nylon. This is important, in that the subsequent detection steps require as much of the possible analyte, or target biomolecule, to be available for (in a DNA detection example) hybridization with the labeled probe. Following the immobilization, there are typically several liquid immersion steps including blocking, washing, hybridization buffer exposure, etc. Each step has the potential of removing analyte from the glass surface, and decreasing the potential strength of the signal. Nylon is generally regarded as having the highest biomolecule binding efficiency when compared to other the commercially available polymer or other treated substrates. Nylon is also regarded as providing highest accessibility of the functional groups of the analyte thus bound to the nylon surfaces.
Nylon membranes, a specific species of microporous membrane, formed by a phase inversion process, have some advantages over nitrocellulose membranes in that nylon is naturally hydrophilic. Nylon membranes also have a greater protein and DNA binding capacity than nitrocellulose. This increased binding capacity means better signal strength and lower detection thresholds in assays.
Nylon membrane pore structure is more easily controllable than nitrocellulose membrane pore structure and is more physically robust than the nitrocellulose membranes. Nitrocellulose is more brittle than the nylon membrane, has more pore variability and is extremely flammable. The physical weakness, variability and flammability of the nitrocellulose membranes combine to make nitrocellulose membrane more expensive to manufacture than nylon membrane.
As discussed above, there are at least three main disadvantages to scrim-reinforced nylon glued or otherwise adhered to a glass substrate. First, the glue or adhesive layer adds additional thickness to the combination scrim-reinforced nylon/glass slide. The arraying robots that blot the nylon membranes have narrow spatial tolerances, and any additional thickness represents additional uncertainty about accurate positioning of the combination scrim-reinforced nylon/glass slide relative to the arraying robots. The second, and more important, disadvantage is that the scrim-reinforced membrane on the combination scrim-reinforced nylon/glass slide has an irregular surface on the micro scale. This is an important cosmetic problem since the spot sizes made on the membrane are on a similar scale. Finally, the glue/adhesive and the analyte may not be compatible. Specifically, the adhesive which contains an excess of functionalized moieties for attachment can indiscriminatiely bind the analyte in a way which makes it unavailable for detection; either by binding to the molecule preventing (in the DNA example) hybridization, or by reversibly binding to the analyte such that the attachment is not permanent, and the analyte is sloughed off in the liquid immersion steps prior to detection. Finally, the adhesive itself can be degraded in the multi-step processes leading to detection, and become, by extraction or other means, a mobile species. The adhesive fragment, if bound to the analyte, may be displaced to a location or area beyond the location of detection, or itself become part of a false background signal, depending on the type of detection being performed.
In these types of microarray slides, it is useful to have a nylon microporous layer that is flat, uniform, and is as thin as possible. In the case of charge modified slides, the degree of charge modification must be uniform over the entire slide surface. In the environment of use, as envisioned for the innovative slide""s described in the present application, the bond between the nylon and the base member, such as, for example, a glass slide or Mylar sheet, must remain stable in water, NaOH, sodium dodecyl sulfate, and other harsh chemicals for prolonged periods of time and at high temperatures. Because of the high air pressure generated between the nylon membrane layer and the glass substrate when the nylon membrane is wetted, the bond therebetween must also be physically strong.
Further, it would be desirable to use fluorescent assays, as opposed to isotopic assays, if the detection sensitivity of fluorescent assays could be enhanced without increasing the potential for undesired chemical reactions. While sensitivity can be increased if the substrate on which fluorescent assays are performed does not fluoresce upon such exposure, isolation of such substrates having widespread usefulness (with respect to numerous analytes) has so far eluded the art.
Specifically, great varieties of assay systems have been developed to detect the presence and concentration of analytes in samples. For example, bioaffinity and enzymatically activated catalysis reactions are widely used in medicine and science to analyze biological samples to detect and quantitize biological materials of concern. Many of these assay systems depend upon the binding of one chemical entity with the material of concern (or a modified form thereof) and detection of the conjugate, e.g., antigen-antibody, nucleic acid strand to complementary nucleic acid strand (xe2x80x9chybridizationxe2x80x9d), and protein-ligand conjugates. The conjugate is typically detected by way of a label providing a detectable signal that is attached to one or more of the binding materials. The conjugate is frequently quantitated by first determining the amount of label in the free and bound fractions, and then calculating the amount present using an algorithm and a set of standards to which the samples are compared.
The most common labels used in analyte binding assays are radioisotopes and luminescent compounds. Luminescence is induced by energy transfer and refers to light emission that cannot be attributed merely to the temperature of the emitting body. Luminescent labels can be made to luminesce through photochemical (so-called, xe2x80x9cphotoluminescencexe2x80x9d), chemical (so-called, xe2x80x9cchemiluminescencexe2x80x9d) and electrochemical (so-called, xe2x80x9celectrochemiluminescencexe2x80x9d) means. Photoluminescence, which includes fluorescence and phosphoresence, is a process whereby a material is induced to luminesce when it absorbs electromagnetic radiation such as visible, infrared or ultraviolet radiation. Chemiluminescence refers to luminescence occurring as a result of a chemical reaction without an apparent change in temperature. Electrochemiluminescence refers to luminescence occurring as a result of electrochemical processes.
Isotopic labeling proffers considerably better detection in certain analyte systems than luminescent labeling. For example, the most sensitive methods for detecting nucleic acids typically involve the use of isotopic labeling, often involving radiolabelling with 32P.
In localizing particular sequences within genomic deoxyribonucleic acid (xe2x80x9cDNAxe2x80x9d), a transfer technique described by Southern is typically employed. DNA is digested, often using one or more restriction enzymes, and the resulting fragments are separated according to size by electrophoresis through a gel. Conventionally the DNA is then denatured in situ and transferred from the gel to a solid support, the relative positions of the DNA fragments being preserved during and after the transfer to the solid support. The DNA attached to the solid support is then hybridized to radiolabelled DNA or ribonucleic acid (xe2x80x9cRNAxe2x80x9d), and autoradiography is used to locate the positions of bands complementary to the probe.
For many years, immobilization and hybridization of denatured DNA was carried out almost exclusively using nitrocellulose as a solid support. As time progressed, however, it became apparent that nitrocellulose was a less than an ideal solid-phase hybridization matrix, as nucleic acids are attached to the nitrocellulose support by hydrophobic, rather than by covalent, interactions, and the nucleic acids are released slowly from the matrix during hybridization and washing at high temperatures. To overcome this problem, charge-modified cellulose supports, including DBM(diazobenzyloxymethyl)-cellulose and APT-cellulose, were introduced in the early 1980""s to provide improved nucleic acid binding. These matrices however, like nitrocellulose itself, also suffer from a significant disadvantage in that they become brittle when dry and cannot survive more than one or two cycles of hybridization and washing, i.e., xe2x80x9creprobing.xe2x80x9d
Extensive use today is made of polyamide matrices, in particular nylon matrices, as solid support for immobilization and hybridization of nucleic acids. Various types of nylon are known to bind nucleic acids irreversibly and are far more durable than nitrocellulose. As nucleic acids can be immobilized on nylon in buffers of low ionic strength, transfer of nucleic acids from gels to a nylon matrix can be carried out electrophoretically, which may be performed if transfer of DNA by capillary action or vacuum is inefficient. Two basic types of nylon membranes are commercially available, unmodified nylon and charge-modified nylon. Charge-modified nylon is preferred for transfer and hybridization as its increased positively charged surface has a greater capacity for binding nucleic acids (See, e.g., U.S. Pat. No. 4,473,474, the disclosure of which is herein incorporated in its entirety by reference). Nylon membranes must be treated to immobilize the DNA after it has been transferred, as by way of thorough-drying, or exposure to low amounts of ultraviolet radiation (254 nm).
While polyamide matrices have found considerable use in isotopic assay systems, such matrices have not found widespread use in fluorescent assay systems. This is likely because fluorescent assay systems employing polyamide substrates demonstrate less than desirable sensitivity. Such reduction in sensitivity has been attributed primarily to two factorsxe2x80x94background fluorescence produced by the nylon itself, and light scattering by solid materials in contact with the reaction media (such as substrates to which reactants are attached, or walls of the containers in which measurements are made). Polyamides, such as nylon, show light-stimulated endogenous fluorescent emissions and light reflection which can coincide with the range of UV-visible wavelengths emitted from fluorophore-tagged analytes. When light in the excitation waveband causes fluorescence of the support material, interference with detection occurs if the emission waveband of the fluorophore overlaps the same.
While isotopic assays, overall, are very sensitive, they suffer from a number of disadvantages. Primarily, use of any radioisotope automatically invokes health concerns and a host of regulatory duties with respect to waste disposal, safety, handling, reporting and licensing. While present luminescent assays proffer an alternative to isotopic labeling, the sensitivity of such assays is still not within a range desired by many in the biomedical, genetic research and drug discovery communities. Additionally, isotopic labeling cannot be used in multiplex assays, in which two or more nucleic acid probes which have been separately labeled each with their own unique wavelength-emitting luminescent molecule can be simultaneously hybridized, then simultaneously detected on an array of bound nucleic acid targets affixed to the polymeric substrate. Multiplexing saves significant cost and time when compared to the traditional steps of stripping and reprobing when performing multiple queries on a given array of targets. Multiplexing also reduces error and signal degradation that is associated with multiple reprobings.
Thus, there is a need for a relatively thin, multi-cell non-luminescent substrate useful for Micro-Analytical Diagnostic Applications. Such composite microarray non-luminescent slides"" structure should be naturally hydrophilic. Such composite microarray non-luminescent slides"" properties should be easily controlled. Such composite microarray non-luminescent slides should be more physically robust than the nitrocellulose membrane slides of the prior art. Such composite microarray non-luminescent slides should be relatively easily manufactured. Such composite microarray non-luminescent slides should at least minimize, if not eliminate, any glue/adhesive layer between the membrane and the solid substrate which adds thickness to the membrane/substrate combinations. Such composite microarray non-luminescent slides should have a surface treatment for a substrate that prepares the substrate to operatively, covalently bond to la microporous membrane formed by a phase inversion process such that the combination produced thereby is useful in microarray applications. Such composite microarray non-luminescent slides should include a surface treatment that has no discernable finite thickness or mass which could add nonuniformity to the overall thickness of the composite microarray slides having a porous membrane formed by a phase inversion process useful in microarray applications. Such composite microarray non-luminescent slides should include a surface treatment that at least minimizes, if not eliminates, the participation of this treatment in the binding or detection of nucleic acid or protein analytes by a composite microarray slides having a porous membrane formed by a phase inversion process useful in microarray applications. Such composite microarray non-luminescent slides should include a porous membrane formed by a phase inversion process useful in microarray applications which includes a surface treatment to the solid substrate that minimizes the interference of the substances used to connect the solid substrate portion to the porous membrane portion used for the detection of analytes. Such composite microarray cell non-luminescent slides should include a porous membrane formed by a phase inversion process useful in microarray applications which includes a surface treatment that eliminates nonuniformity of the overall thickness of the substrate/membrane combination structure which is associated with using a third component having a finite thickness or mass as the connecting agent. Such composite microarray non-luminescent slides should have a regular surface on the micro scale. Such composite microarray non-luminescent slides should eliminate compatibility issues between the glue/adhesive and the analyte. Such composite microarray slides should be economically produced. Such composite microarray non-luminescent slides should be for use in luminescent assays which lead to greater sensitivity for detecting analytes in a sample. Such composite microarray slides should allow for simultaneous use of different fluorescently labeled tags for simultaneous detection of multiple analyte molecules.
An object of the present disclosure is to provide composite microarray non-luminescent slides having a surface treatment for a substrate that prepares the substrate to operatively, covalently bond to a microporous membrane formed by a phase inversion process such that the combination produced thereby is useful in microarray applications.
Another object of the present disclosure is to provide a surface treatment that has no discernable finite thickness or mass which could add nonuniformity to the overall thickness of composite microarray non-luminescent slides having a porous membrane formed by a phase inversion process useful in microarray applications.
A further object of the present disclosure is to provide a surface treatment that minimizes participation in the binding or detection of nucleic acid or protein analytes of composite microarray non-luminescent slides having a porous membrane formed by a phase inversion process useful in microarray applications.
Yet a further object of the present disclosure is to provide composite microarray non-luminescent slides having a porous membrane formed by a phase inversion process useful in microarray applications which includes a surface treatment that minimizes the interference of the substances used to connect the solid substrate portion to the porous membrane portion thereof with the detection of analytes.
Yet another object of the present disclosure is to provide a method for fabricating composite microarray non-luminescent slides having a surface treatment for a substrate that prepares the substrate to sufficiently, covalently bond to a microporous membrane formed by a phase inversion process such that the combination produced thereby is useful in microarray applications.
Still another object of the present disclosure is to provide composite microarray non-luminescent slides having a porous membrane formed by a phase inversion process useful in microarray applications which includes a surface treatment that eliminates nonuniformity of the overall thickness of the substrate/membrane combination structure which is associated with using a third component having a finite thickness or mass as the connecting agent.
In accordance with these and further objects, one aspect of the present disclosure includes a method of fabricating non-luminescent composite microarray slides useful for carrying a microarray of biological polymers comprising the acts of: providing a non-porous substrate; providing a non-luminescent microporous membrane formed by a phase inversion process, the process comprising the acts of: formulating a dope comprising a solvent, one or more non-solvents, opaque solids, and polyamide(s); mixing the dope to cause dissolution of the polyamide and opaque solids therein; producing an opaque solids-filled phase inversion dope; casting a portion of the opaque solids-filled phase inversion dope; and quenching the cast portion of the opaque solids-filled phase inversion dope to form a non-luminescent, microporous membrane; providing a surface treatment; applying the surface treatment to the non-porous substrate; and intermingling the non-porous substrate having the surface treatment with the non-luminescent, microporous membrane such that the non-porous substrate is sufficiently covalently bonded to the non-luminescent microporous membrane wherein the combination produced thereby is useful in microarray applications.
Another aspect of the present disclosure includes composite microarray slides useful for carrying a microarray of biological polymers comprising: a substantially non-reflective microporous membrane which provides little fluorescence from about three hundred (300) nm to about seven hundred (700) nm formed by a phase inversion process, the non-reflective microporous membrane comprising: a phase-inversion support; and a plurality of opaque solids that are substantially chemically non-reactive with the phase inversion support and intimately bound to, and/or partially/completely contained within, said phase-inversion; a non-porous substrate; and a surface treatment, operatively positioned between the substantially non-reflective microporous membrane and the non-porous substrate, for sufficiently covalently bonding the non-porous substrate to the microporous membrane wherein the combination composite microarray slides produced thereby are useful in microarray applications.
A third aspect of the present disclosure includes composite microarray slides useful for carrying a microarray of biological polymers comprising: an optically passive substrate comprising: a phase-inversion support and opaque solids that are substantially non-reactive chemically with the phase-inversion support, in a weight ratio with the phase-inversion support such that they optically passive substrate absorbs light at substantially all wave lengths from about three hundred (300) nm to about seven hundred (700) nm; a non-porous substrate; and a surface treatment, operatively positioned between the optically passive substrate and the non-porous substrate, for sufficiently covalently bonding the non-porous substrate to the optically passive substrate wherein the, combination microarray slides produced thereby are useful in microarray applications.
Another aspect of the present disclosure may include a post-treatment of the non-luminescent microporous membrane such that the membrane contains a greater positive charge; such a treatment is useful in augmenting the microporous membrane""s ability to retain biological polymers, which predominantly are negatively charged.
Other objects and advantages of the disclosure will be apparent from the following description, the accompanying drawings and the appended claims.